DNA replication is strictly regulated to allow only one round per cell cycle. Over-replication can lead to serious consequences including genomic instability, cancer, or death. Thus, levels of the Cdt1/Dup replication licensing factor are tightly regulated. Cdt1/Dup helps load the replicative helicase complex onto origins of replication, and its proper expression and timely destruction are an important part of the "once and only" replication control during S phase. Significantly, elevated levels of Cdt1/Dup and its inhibitor Geminin are associated with an increasing number of human cancers. Recent reports indicate that members of the L2DTL family play a vital role in maintaining genomic stability by modulating Cdt1/Dup levels immediately after S phase entry. In particular, Lethal 2 Denticleless (L2DTL) acts as a specificity factor, targeting Cdt1/Dup to the E3 ubiquitin ligase containing Cullin 4 (Cul4) and DNA damage binding factor 1 (DDB1) for subsequent proteasome degradation. Increased levels of L2DTL itself are associated with hepatocellular carcinomas: the liver being one of the few adult tissues sustaining a re-replicative cell cycle. Notably, Drosophila studies highlight the role of Cdt/Dup in tissues undergoing re-replicative cycles. Since Cdt1/Dup has been extensively studied in Drosophila in various cell and development contexts, Drosophila melanogaster serves as an excellent model for elucidating the role of the L2DTL family in regulating Cdt1/Dup levels and maintaining genomic stability. To determine the function of L2DTL in a multicellular organism, this proposal will focus on identifying and characterizing fly stocks with mutations in the l(2)dtl gene. Characterization will include an assessment of the re-replication phenotype in embryonic tissues using BrdU incorporation analysis and evaluation of Cdt1/Dup levels using indirect immunofluorescence (specific aim 1). Further, anti-L2DTL antibodies will be generated to investigate the L2DTL protein expression pattern and subcellular localization in cells undergoing mitotic and endocyclic cell cycles. Finally, since mutation of the l(2)dtl gene results in embryonic lethality, Drosophila lines for inducing mitotic clones in larval and adult tissues will be generated to elucidate the role of L2DTL in maintaining genomic stability in somatic tissues as measured by BrdU incorporation and Cdt1/Dup accumulation (specific aim 2). Experiment results should support the hypothesis that L2DTL is essential for preventing re-replication in all cell contexts.